Repeated influenza vaccination induces similar immune protection as first‐time vaccination but with differing immune responses

Abstract Background Recent seasonal epidemics of influenza have been caused by human influenza A viruses of the H1N1 and H3N2 subtypes and influenza B viruses. Annual vaccination is recommended to prevent infection; however, how annual influenza vaccination influences vaccine effectiveness is largely unknown. Methods To investigate the impact of repeated vaccination on immune and protective effect, we performed a prospective seroepidemiologic study. Participants with or without prior vaccination (2018–2019) were enrolled during the 2019–2020 influenza season. Inactivated quadrivalent influenza vaccine (IIV4) was administered through the intramuscular route, and venous blood samples were collected regularly to test hemagglutination inhibition (HAI) titers. Results The geometric mean titers and proportion with titers ≥40 against the influenza vaccine components peaked at 30 days post‐vaccination. At Day 30, the geometric mean titer and proportion with titers ≥40 in participants who had been previously vaccinated were higher for H3N2 but similar for both B lineages (Victoria and Yamagata) as compared with participants vaccinated for the first time. As for H1N1, the geometric mean titer was lower in repeated vaccinated participants, but the proportion with titers ≥40 was consistent in both groups. Conclusions Repeated vaccination provides similar or enhanced protection as compared with single vaccination in first‐time vaccinees.

geometric mean titer and proportion with titers ≥40 in participants who had been previously vaccinated were higher for H3N2 but similar for both B lineages (Victoria and Yamagata) as compared with participants vaccinated for the first time. As for H1N1, the geometric mean titer was lower in repeated vaccinated participants, but the proportion with titers ≥40 was consistent in both groups. Since 2010, the Advisory Committee on Immunization Practices has recommended annual influenza vaccination for all persons aged ≥6 months who do not have contraindications. 7 However, this recommendation has faced the question of how prior vaccination may influence the immune response to the current vaccine. Previous researches have suggested that repeated annual influenza vaccination can blunt future vaccine-induced antibody responses, particularly to IAV H3N2 strains, [8][9][10][11] raising the concern that frequent vaccination may impair vaccine immunogenicity and effectiveness. On the contrary, other studies of influenza vaccines indicated that residual protection against influenza viruses occurred even though the predominant viruses were antigenically distinct from the previous season's vaccine components. 12,13 In this study, we compared antibodies characterized by hemagglutination inhibition (HAI) titer in individuals vaccinated for two consecutive years with those who received vaccine in the current season only, evaluating whether vaccination history influences antibody response induced by inactivated, quadrivalent split-virion influenza vaccine (IIV4). This study was a prospective observational cohort, designed to examine the similarities and differences in the humoral immune response between individuals with different influenza vaccination histories.

| Participant enrollment
Subjects were enrolled using random cluster sampling. 14

| Sample collection
Pre-vaccination serum samples were collected on December 9, 2019, and then the IIV4 injections were administered on the same day.
Post-vaccination sera were collected at 7, 30, and 245 days after vaccination. Samples were aliquoted and frozen at À80 C until assayed.
Participants were instructed to report influenza-like illness (ILI). Participants who had an axillary temperature of 37.8 C or higher and at least one respiratory (cough, pharyngitis, or dyspnea) or systemic (chills, headache, malaise, or myalgia) symptom were asked to selfreport. If ILI was reported, a throat swab was taken within 24 h after onset of symptoms, which was used for influenza virus detection and subsequent subtyping of IAV and IBV by real-time Reverse Transcription Polymerase Chain Reacation (RT-PCR).

| Antigenic components
All participants received a single 0.5 ml intramuscular injection of the quadrivalent vaccine for 2019-2020 season in the deltoid muscle.  for HAI testing were stored in multiple aliquots at À80 C. HAI assays were performed with pre-and post-vaccination serum specimens using 1% turkey erythrocytes. Sera treated with receptor destroying enzyme and adsorbed on guinea pig or chicken red blood cells before the test were diluted twofold starting from a 1:10 dilution. HAI titers were defined as the highest serum dilution that completely inhibited hemagglutination and were recorded as 1:5 when below 1:10. 15

| Outcomes measures
The outcomes were serologic responses to egg-grown vaccine refer- is generally accepted to correspond to a 50% reduction in the risk of contracting influenza in a susceptible population. 19,20 The proportion of participants with titers ≥1:40 was used as another outcome measure.

| Statistical analysis
Statistical analysis was performed using IBM SPSS version 24. Statistical significance was assumed when p < 0.05. Statistical tests included Student's t test for continuous variables, non-parametric tests for discrete variables and skewed distribution data, and chi-square test for categorical variables. Data analyzed in this paper are available from the corresponding author on reasonable request.

| GMTs of antibodies after vaccination
The immune response profile following vaccination was analyzed by HAI test in pre-and post-vaccination sera of the volunteers. Dynamic changes in immunogenicity could be observed among the vaccinees before and after vaccination during the study period in 2019-2020 ( Figure 2 and Table 1). Before vaccination, the GMT in the prior vaccination group was higher than that in the single vaccination group for each influenza vaccine component. Although IIV3 in 2018-2019 did not cover B/Yamagata lineage, the GMT against B/Yamagata was still statistically higher in the prior vaccination group before vaccination in 2019-2020 (p = 0.017). Serum HAI antibodies to each of the four vaccine strains in both groups increased rapidly after vaccination and then decreased again toward the end of the influenza season. For every vaccine strain, Mauchly's test of sphericity indicated that there was a remarkable difference in the HAI titers among four sampling times. It also indicated that the GMT trends from pre-vaccination to post-vaccination were significantly different between the prior F I G U R E 1 Monitoring flow chart. One hundred and ninety-three participants were recruited for this study. Pre-vaccination sera were collected on December 9, 2019, and the IIV4s injections were administered on the same day (T 0 ). Post-vaccination sera were collected at 7 (T 1 ), 30 (T 2 ), and 245 (T 3 ) days after vaccination. Three participants in the single vaccination group dropped out before the fourth blood sample was collected. Abbreviation: IIV3, inactivated, trivalent split-virion influenza vaccine; IIV4, inactivated, quadrivalent split-virion influenza vaccine

| Immune protection
To further evaluate the efficacy of seasonal influenza vaccines, we compared SCRs and the proportion with titers ≥40 between the two vaccination groups. SCRs against A/H1N1 were lower in the prior vaccination group than in the single vaccination group, and the differences were statistically significant (48.89% vs 84.47%, p < 0.001).
SCRs post-vaccination against A/H3N2, B/Victoria, and B/Yamagata were largely consistent between the two groups ( Table 2).
Given that the seroconversion influenced by the baseline GMT and the GMTs among participants in the prior vaccination group was significantly higher than the other group, we compare the percentage of vaccinees achieving HAI titers ≥40 in both groups, which is generally accepted to correspond to a 50% reduction in the risk of contracting influenza in a susceptible population. 19,24 Baseline proportion with titer ≥40 in the prior vaccination group was higher than that in the single vaccination group with significant differences for A/H1N1 (54.44% vs 26.21%, p < 0.001), A/H3N2 (17.78% vs 7.77%, p = 0.036), and B/Victoria (11.11% vs 0.00%, p = 0.002). Of note, baseline proportions with titer ≥40 for A/H3N2, B/Victoria, and B/Yamagata in both groups were consistently lower than that for A/H1N1. For all four vaccine viruses, the proportion with titer ≥40 increased rapidly after vaccination and peaked on Day 30, but the rates of increase were different ( Table 3). The percentage of participants with an HAI titer ≥40 for the IAVs increased more rapidly compared with IBVs. At the peak, the percentage increased to more than 90% among all participants for A/H1N1, about 80% for A/H3N2, 50% for B/Victoria, and more than 60% for B/Yamagata. Thereafter, the proportion decreased as GMT declined. For A/H1N1 and A/H3N2, the percentage of participants with an HAI titer ≥40 remained higher than 80% at Day 245, although the GMT was significantly lower than at the peak. However, there were significant T A B L E 1 The geometric mean titers pre-and post-vaccination for individuals in Jiangsu province, 2019-2020 antibody levels increased rapidly after vaccination, and there was a The antigenic distance hypothesis is one potential explanation, which suggests that repeat vaccine efficacy can be influenced by antigenic distances among the influenza vaccines of previous and current seasons, and the current epidemic strain. 37,38 Sequence differences in influenza vaccine strains between two consecutive seasons could be one of the reasons for the discrepancy in these findings. Additional research on cross-reactivity of antibodies and/or T-cells between antigenically distinct viruses will be beneficial to understanding the mechanism behind whether pre-existing immunity boosts or impairs reactive responses to the current influenza vaccine.
Actually, the concept of seroprotection can be defined as titer ≥40 within the vaccines as used in previous studies, [39][40][41] although it is also indicated that titer ≥40 can only contribute 50% protection. 19,24 Herein, in our study, 25 participants (

CONFLICT OF INTEREST
The authors declare no competing interests.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13060.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.